We would like to thank Marc Leonard from Carleton Immersive Media Studio for expert technical assistance in video production and editing and Dr. Martin Bertrand from Carleton Immersive Media Studio / Neural Regeneration Laboratory for visual model assistance. The authors gratefully acknowledge the expert advice of Dr. Marilyn Keaney and all of her dedicated staff at the University of Ottawa Animal Care and Veterinary Services. This work was funded by the Canadian Institute of Health Research (CIHR, MOP 62826) to SALB, the CIHR Institute of Aging and Strategic Training Initiative in Health Research/CIHR Training Program in Neurodegenerative Lipidomics (TGF 96121) to SALB and SF, Canadian Foundation for Innovation to SF, Ontario Innovation Trust to SF, and Autodesk Research to SF. ACM receives a CIHR Banting and Best doctoral award. NV receives a post-professional fellowship from Institute of Aging and CIHR Training Program in Neurodegenerative Lipidomics.

1. Preparing Reagents 
<ol>
 <li>For sterile vaginal lavage, autoclave double distilled water (ddH2O) and store in a tightly sealed container at room temperature until needed. </li>
 <li>For cytological assessment, add 0.1 g of crystal violet powder to 100 ml of ddH2O. Mix well. Crystal violet stain (0.1%) can be stored in a tightly sealed container at room temperature until needed. </li>
</ol>
2. Collecting Vaginal Cells (Vaginal Lavage)
<ol>
 <li>Place a latex bulb on the end of a sterile 200 μl tip and draw up approximately 100 μl of sterile ddH2O using the gradations on the tip as a volume guideline. </li>
 <li>Lift the mouse out of her cage and place her on the cage hopper (lid) with her hind/rear end towards you. </li>
 <li>Firmly grasp the tail and elevate the rear end. The mouse will now have only her front paws grasping the hopper. At this point the mouse may urinate. If so, wait until urination stops. Should there be urine left at the entrance to the vaginal canal, you may want to rinse the opening with excess ddH2O using a separate tip (i.e., not your sample collection tip).</li>
 <li>Place the end of the ddH2O-filled tip at the opening of the vaginal canal taking care to not penetrate the orifice as vaginal (and cervical) stimulation can induce pseudopregnancy in rats1,2. Recent reports suggest mice are less susceptible to this effect nonetheless care should be taken to minimize the degree of invasiveness in repeated analyses3. </li>
 <li>Gently depress the bulb to expel a quarter to half of the volume of water (~25-50 μl) at the opening of vaginal canal. The liquid will spontaneously aspirate into the canal without tip insertion. Slowly release the pressure exerted on the bulb. The fluid will withdraw back into the tip. Avoid releasing pressure too quickly to prevent aspiration of fluid into the bulb. A filtered tip may be useful for this purpose. </li>
 <li>Repeat the previous step 4-5 times using the same tip, bulb, and fluid to obtain a sufficient number of cells in a single sample. </li>
 <li>Place the fluid on glass slide, and allow the smear to completely dry at room temperature. Once dry, these estrous smears can be stained immediately or stored and stained at a later date. </li>
</ol>
3. Cytological Staining using Crystal Violet*4
<ol>
 <li>Place the dry slide in a coplin jar (or other comparable staining vessel) containing the crystal violet stain for 1 min. </li>
 <li>Remove to a second coplin jar containing ddH2O. Wash the slide with ddH2O for 1 min. Repeat. </li>
 <li>Remove the excess ddH2O from the edges of the slide with a light-duty tissue wiper, avoiding contact with the stained smear.</li>
 <li>Pipette approximately 15 μl of glycerol on top of the smear and coverslip. Alternatively, other histological mounting reagents can be utilized to obtain a more permanent, non-diffusing stain. </li>
</ol>
 * The staining method described here is the simplest procedure that can be performed in any laboratory. Other methods can provide additional details. For example, using Papanicolaou staining, the maturity of nucleated epithelial cells can be distinguished with less mature cells stained turquoise and more mature cells pink- or orange-stained. These differences can be used to stage early or late proestrus.4 
4. Vaginal Cytology
<ol>
 <li>Examine the smear under light microscopy to determine cell types present. Microscopic examination should be done immediately after staining as the crystal violet will diffuse from the cells over time when using glycerol for coverslipping. Photomicrographs should be taken at time of analysis to document cytology.</li>
 <li>Start by examining the entire smear at a lower magnification. Select a representative area and move to a higher magnification. You will see cornified squamous epithelial cells, leukocytes, and/or nucleated epithelial cells (representative results, Figure 1A-C). The ratio of cells present will allow you to determine the estrous stage of your mouse at time of sample collection (representative results, Figure 1D-G) and her immediate hormonal status (discussion, Figure 2). </li>
</ol>
5. Representative Results
Cytology: Three primary cell types can be detected in vaginal smear samples: (1) nucleated epithelial cells (Figure 1A), (2) cornified squamous epithelial cells (Figure 1B), and (3) leukocytes (Figure 1C). Nucleated epithelial cells have a lightly stained cytoplasm, darker stained plasma membrane, and an oval nucleus (Figure 1A). Cornified squamous epithelial cells are uniformly stained, more polygonal in shape than their nucleated epithelial predecessors, and lack a nucleus (Figure 1B). Polymorphonuclear leukocytes can be distinguished from epithelial cells by their irregular shape, darkly stained polymorphic nuclei, and small size (Figure 1C, black arrows). Should urine contamination be present in the smear, uric acid crystals are readily detected by their crystalline structures dissimilar to any expected cell types (Figure 3). Should this occur, and obscure detection of predominant cell type, the smear should be discarded and not used for staging purposes.
Staging: The relative ratio of cell types observed in smears can be used to identify the stage of the estrous cycle of your mouse on the day of sample collection (Figure 1D-G). During proestrus, cells are almost exclusively clusters of round, well-formed nucleated epithelial cells (Figure 1D, representative cell indicated by white arrow). During estrus, cells are predominantly cornified squamous epithelial cells, present in densely packed clusters (Figure 1E, representative cell indicated by arrowhead). During metestrus, small darkly stained leukocytes predominate (Figure 1F, representative cell indicated by black arrow). Cornified squamous epithelial cells may be observed, often in fragments, (Figure 1F, representative cell indicated by black arrowhead). During diestrus, rare cornified squamous epithelial cells may still be present (Figure 1G, representative cell indicated by black arrowhead), however leukocytes still predominate (Figure 1G, representative cell indicated by black arrow). Metestrus can be distinguished from diestrus by the appearance of nucleated epithelial cells in diestrus (Figure 1G, representative cell indicated by white arrow). 
<img src="/files/ftp_upload/4389/4389fig1.jpg" alt="Estrous cycle microscopy images; nucleated, cornified cells, leukocytes; proestrus, estrus stages." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Cytological assessment of vaginal smears can be used to identify estrous stage. Three main cell types are detected in vaginal smear samples: (A) nucleated epithelial cells, (B) cornified squamous epithelial cells, and (C) leukocytes. The ratio of these cell types present in the smear can be used to identify mice in (D) proestrus, (E) estrus, (F) metestrus, or (G) diestrus as described in representative results. Black arrowheads in E, F and G point to representative cornified squamous epithelial cells. Black arrows in C, F and G point to representative leykocytes. White arrows in D and G highlight representative nucleated epithelial cells.
<img src="/files/ftp_upload/4389/4389fig2.jpg" alt="Hormone level chart and cell micrographs depicting estrous cycle phases: proestrus, estrus, metestrus, diestrus." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Vaginal smear cytology reflects underlying endocrine events. Details are also provided in Discussion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4389/4389fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/4389/4389fig3.jpg" alt="Microscope image of crystallization process, arrows pointing to crystals, highlighting morphology." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Uric acid crystals may be present following crystal violet staining of urine-contaminated samples. (A) Crystals are transparent and can be of various sizes (arrows and boxed region magnified in (B)). No cells are present in this field. Should uric acid crystal contamination within fields used for cytological staining be present, it may be difficult to accurately identify cell types present and the smear should be discarded. Scale bars = 50 μm.

<ol>
	<li>Adler, N. T., Zoloth, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copulatory+behavior+can+inhibit+pregnancy+in+female+rats.">Copulatory behavior can inhibit pregnancy in female rats.</a> Science. 168, 1480-1482 (1970).</li><li>Adler, N. T., Resko, J. A., Goy, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+copulatory+behavior+on+hormonal+change+in+the+female+rat+prior+to+implantation.">The effect of copulatory behavior on hormonal change in the female rat prior to implantation.</a> Physiology. 5, 1003-1007 (1970).</li><li>Yang, J. J., Larsen, C. M., Grattan, D. R., Erskine, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating-induced+neuroendocrine+responses+during+pseudopregnancy+in+the+female+mouse.">Mating-induced neuroendocrine responses during pseudopregnancy in the female mouse.</a> Journal of. 21, 30-39 (2009).</li><li>Hong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+mouse+estrus+cycle+in+response+to+BRCA1+inactivation+suggest+a+potential+link+between+risk+factors+for+familial+and+sporadic+ovarian+cancer.">Changes in the mouse estrus cycle in response to BRCA1 inactivation suggest a potential link between risk factors for familial and sporadic ovarian cancer.</a> Cancer research. 70, 221-228 (2010).</li><li>Hawkins, S. M., Matzuk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+menstrual+cycle:+basic+biology.">The menstrual cycle: basic biology.</a> Annals of the New York Academy of Sciences. 1135, 10-18 (2008).</li><li>Walmer, D. K., Wrona, M. A., Hughes, C. L., Nelson, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+expression+in+the+mouse+reproductive+tract+during+the+natural+estrous+cycle:+correlation+with+circulating+estradiol+and+progesterone.">Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone.</a> Endocrinology. 131, 1458-1466 (1992).</li><li>Parkening, T. A., Collins, T. J., Smith, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+pituitary+concentrations+of+LH,+FSH,+and+prolactin+in+aging+C57BL/6+mice+at+various+times+of+the+estrous+cycle.">Plasma and pituitary concentrations of LH, FSH, and prolactin in aging C57BL/6 mice at various times of the estrous cycle.</a> Neurobiology of aging. 3, 31-35 (1982).</li><li>Sarkar, D. K., Chiappa, S. A., Fink, G., Sherwood, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropin-releasing+hormone+surge+in+pro-oestrous+rats.">Gonadotropin-releasing hormone surge in pro-oestrous rats.</a> Nature. 264, 461-463 (1976).</li><li>Rajendren, G., Gibson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+confocal+microscopic+study+of+synaptic+inputs+to+gonadotropin-releasing+hormone+cells+in+mouse+brain:+regional+differences+and+enhancement+by+estrogen.">A confocal microscopic study of synaptic inputs to gonadotropin-releasing hormone cells in mouse brain: regional differences and enhancement by estrogen.</a> Neuroendocrinology. 73, 84-90 (2001).</li><li>Kumar, T. R., Wang, Y., Lu, N., Matzuk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+stimulating+hormone+is+required+for+ovarian+follicle+maturation+but+not+male+fertility.">Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility.</a> Nature. 15, 201-204 (1997).</li><li>Montgomery, V., Loutradis, D., Tulchinsky, D., Kiessling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSH-induced+ovulation+in+intact+and+hypophysectomized+mice.">FSH-induced ovulation in intact and hypophysectomized mice.</a> Journal of reproduction and fertility. 84, 1-6 (1988).</li><li>Mihm, M., Gangooly, S., Muttukrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+normal+menstrual+cycle+in+women.">The normal menstrual cycle in women.</a> Animal reproduction science. , 124-229 (2011).</li><li>Appelgren, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+demonstration+of+drug+interference+with+progesterone+synthesis.">Histochemical demonstration of drug interference with progesterone synthesis.</a> Journal of reproduction and. 19, 185-186 (1969).</li><li>Sander, V. A., Facorro, G. B., Piehl, L., de Celis Rubin, E., Motta, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+DHEA+and+metformin+on+corpus+luteum+in+mice.">Effect of DHEA and metformin on corpus luteum in mice.</a> Reproduction. 138, 571-579 (2009).</li><li>Stocco, C., Telleria, C., Gibori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+control+of+corpus+luteum+formation,+function,+and+regression.">The molecular control of corpus luteum formation, function, and regression.</a> Endocrine reviews. 28, 117-149 (2007).</li><li>Rudolph, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+overt+menstruation+in+intact+mice.">Induction of overt menstruation in intact mice.</a> PLoS One. 7, e32922 (2012).</li><li>Yano, J., Lilly, E., Barousse, M., Fidel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell-derived+S100+calcium-binding+proteins+as+key+mediators+in+the+hallmark+acute+neutrophil+response+during+Candida+vaginitis.">Epithelial cell-derived S100 calcium-binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis.</a> Infection and immunity. 78, 5126-5137 (2010).</li><li>Whitten, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+anoestrus+in+mice+caged+in+groups.">Occurrence of anoestrus in mice caged in groups.</a> The Journal of endocrinology. 18, 102-107 (1959).</li><li>Lamond, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+stimulation+derived+from+other+animals+of+the+same+species+on+oestrous+cycles+in+mice.">Effect of stimulation derived from other animals of the same species on oestrous cycles in mice.</a> The Journal of endocrinology. 18, 343-349 (1959).</li><li>Nelson, J. F., Felicio, L. S., Randall, P. K., Sims, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+of+estrous+cyclicity+in+aging+C57BL/6J+mice:+I.+Cycle+frequency,+length+and+vaginal+cytology.">A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology.</a> Biology of reproduction. 27, 327-339 (1982).</li><li>Felicio, L. S., Nelson, J. F., Finch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+studies+of+estrous+cyclicity+in+aging+C57BL/6J+mice:+II.+Cessation+of+cyclicity+and+the+duration+of+persistent+vaginal+cornification.">Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification.</a> Biology of reproduction. 31, 446-453 (1984).</li><li>Van Der Lee, S., Boot, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+pseudopregnancy+in+mice.+II.">Spontaneous pseudopregnancy in mice. II.</a> Acta physiologica et pharmacologica Neerlandica. 5, 213-215 (1956).</li><li>Van Der Lee, S., Boot, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+pseudopregnancy+in+mice.">Spontaneous pseudopregnancy in mice.</a> Acta physiologica et pharmacologica Neerlandica. 4, 442-444 (1955).</li><li>Armaiz-Pena, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrous+cycle+modulates+ovarian+carcinoma+growth.">Estrous cycle modulates ovarian carcinoma growth.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 15, 2971-2978 (2009).</li><li>Jemiolo, B., Harvey, S., Novotny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+the+Whitten+effect+in+female+mice+by+synthetic+analogs+of+male+urinary+constituents.">Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents.</a> Proceedings of the National Academy of Sciences of the United States of America. 83, 4576-4579 (1986).</li></ol>

The authors declare no conflict of interest. All experiments on animals were performed in strict accordance with the guidelines and regulations set forth by the University of Ottawa Animal Care Committee and the Canadian Council on Animal Care.

These changes in cell typology are indicative of underlying endocrine events. The proestrus phase of the estrous cycle corresponds to the human follicular phase of the menstrual cycle5 and is defined by a pre-ovulatory increase in circulating 17-&beta;-estradiol levels6, as well as a small surge in prolactin7 (Figure 2, Proestrus, left panel). The increase in 17-&beta;-estradiol indirectly stimulates gonadotropin-releasing hormone neurons in the hypothalamus and septum that, in turn, activate responsive cells in the anterior pituitary to release luteinizing hormone and follicle-stimulating hormone into the circulation8,9 (Figure 2, Proestrus, left panel). In vaginal smears taken from animals in proestrus, cells are almost exclusively oval nucleated epithelial cells (Figure 1D, Figure 2, Proestrus, right panel). The peak in follicle-stimulating hormone levels signals ovulation and entry into estrus10,11. During estrus, 17-&beta;-estradiol levels decline and prolactin levels peak6,7 (Figure 2, Estrus, left panel). Vaginal smears are characterized by almost exclusive detection of irregular-shaped cornified squamous epithelial cells often in clumps (Figure 1E, Figure 2, Estrus, right panel). Entry into metestrus coincides with a continuous rise in progesterone hormone levels6 and corresponds to the beginning of human luteal phase12 (Figure 2, Metestrus, left panel). As progesterone levels start to rise and there is a small surge in 17-&beta;-estradiol levels in response to corpus luteum activation6,13,14 (Figure 2, Metestrus, left panel). The cell types present in vaginal smears during this stage are fragmented, cornified epithelial cells and smaller darker stained leukocytes (Figure 1F, Figure 2, Metestrus, right panel). Finally, entry into diestrus in mice occurs and circulating progesterone levels peak6, corresponding to the human late luteal phase12. Regression of the corpus luteum leads to a subsequent sharp decline in progesterone levels15,16 (Figure 2, Diestrus, left panel). Leukocytes predominate in smears during diestrus. The frequency of cornified epithelial cells is reduced and nucleated epithelial cells begin to be detected just prior to transition to proestrus (Figure 1G, Figure 2, Diestrus, right panel). 
In summary, this simple, routine protocol can be used to estimate daily hormonal fluctuations and establish estrous stage in experimental mice without altering reproductive status if the following precautions are taken. Sampling should be performed no more than once daily using the non-invasive protocol described here as compared to repeated penetration of the vaginal canal, aspiration, and agitation. This can cause vaginal irritation resulting in an inflammatory response17 resulting in leukocytes and other cell types to be present in smears that may confound cytological assessment. Moreover, even in colony-housed females, it is normal to see extended diestrus and estrus stages in different mice as well induction of anestrous18 and this identification is useful in the interpretation of hormonal impact in reproductive, gender, and disease studies. Variability in cycle length is also introduced with age and by housing differences within colonies (individual or group-housing) of females7,19-21. Females housed in female-only colonies can cease cycling and enter a state of prolonged diestrus18,22,23 although cycling can be re-instated by exposure to cages pretreated with male urine to elicit cycling24,25. Thus, to establish individual cycle lengths for a given mouse, it is recommended that the non-invasive assessments described here be performed daily, with care, until two complete cycles are observed.

<table><tbody><tr><td>Sterile 200 &amp;mu;l pipette tips</td><td>Diamed</td><td>E340901</td><td></td></tr><tr><td>Latex bulb (1 ml)</td><td>Fisher</td><td>03-488-21</td><td></td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>Crystal Violet stain (25 g)</td><td>Fisher</td><td>C581-25</td><td></td></tr><tr><td>Light-duty Tissue Wipers</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td></td></tr><tr><td>Microscope Cover Glass (22x30)</td><td>Fisher</td><td>12-544A</td><td></td></tr></tbody></table>

performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

performing-vaginal-lavage-crystal-violet-staining-vaginal-cytological

Nanyang Technological University

Research

JoVE Journal

Biology

112.6K Views.  Neural Regeneration Laboratory and Ottawa Institute of Systems Biology. Hier beschrijven we hoe u de fase van het murine reproductieve (pro-oestrus, oestrus, metestrus of diestrus) door een eenvoudige, niet-invasieve verzamelen en cytologische beoordeling van de vaginale uitstrijkjes te identificeren. Verder hebben we beschrijven hoe vaginale cytologie weerspiegelt circulerende hormoonspiegels ten grondslag liggen aan de transitie door de murine voortplantingscyclus.

Video: Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Immunology and Infection

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Developmental Biology

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Medicine

Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley (SD) adolescent rats. This study demonstrates the complexity of hormonal cycles and the broad spectrum of understanding required to construct a reliable and valid monitoring technique. Through an in-depth examination of principal experimental design and procedural elements,&#160;this description of the cycle and its fundamental principles provides a framework for further understanding and deconstructs misconceptions for future replication.
Along with an outline of the sample collection process employing vaginal lavage, the procedure describes the mechanism of data categorization into the four-stage model of proestrus, estrus, metestrus, and diestrus. These stages are characterized by a new proposed approach, utilizing the 4 categorizing determinants of vaginal fluid condition, cell type(s) present, cell arrangement, and cell quantity at the time of collection. Variations of each stage, favorable and unfavorable samples, the distinction between cyclicity and acyclicity, and graphic depictions of the collected categorizing components are presented alongside effective interpretive and organizational practices of the data. Overall, these tools allow for the publication of quantifiable data ranges for the first time, leading to the standardization of categorization factors upon replication.

This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools.

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley ...

Cervical Manipulation Induced Pseudopregnancy in Mice for Assisted Reproduction

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Author Spotlight: Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination  

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,&#160;female recipient mice must be induced into a pseudopregnant ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Samenvatting

Meer video's verkennen